Over the past 10 years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth and differentiation factor interleukin-2 (IL-2) produced following antigen activation of these cells. Costimulation entails a 30 to 100 fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Clonal anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen-specific receptor. In this case, subsequent re-stimulation of IL-2 production, even in the presence of co-stimulation, is inhibited 10 to 50 fold. Our goals in this project have been to try and understand the molecular mechanisms behind these two phenomena.[unreadable] The prominent affect that costimulation and anergy have on expression of the IL-2 gene has led us to explore its chromatin structure and transcriptional regulation. In FY-2008 we explored the differences in IL-2 gene expression between naive and previously activated T cells. T cell activation with anti-CD3 and anti-CD28 stimulates much more IL-2 mRNA (10 fold) at early time points (4-6 hours) in pre-activated or memory CD4+ T cells than in naive T cells; yet the total amount of IL-2 produced by 72 hours is comparable. To explain this paradox we have now uncovered a biphasic regulation in the transcription of IL-2 in naive T cells. During the first 10 hours after stimulation only 5-10% of naive T cells produce IL-2 in a capture assay. In contrast, greater than 65% of the T cells in a pre-activated population make IL-2 within 6 hours. The same low percentage of naive responding T cells was observed with cells from a GFP Knock-In mouse at the IL-2 locus, showing that there is a limitation in the number of cells transcribing the gene. Beyond 18 hours of stimulation, however, the naive population entered a second phase of IL-2 production which peaked around 48 to 60 hours with over 60% of the cells responding. This late phase expression required cRel, CD28 and TNFR signalling and was completely absent in cells from a double KO lacking both the p55 and the p75 TNF receptors. The late phase expression was correlated with a 10 fold increase in the cell surface expression of the p75 TNF receptor by 24 hours after stimulation. Once pre-activated, however, the remaining T cells no longer required TNFRs for IL-2 production, although they were still partially CD28 and cRel dependent. The quicker response (6 hours) of the pre-activated T cells was associated with a small increase in DNA accessibility at the IL-2 locus in the resting population and a faster and greater increase in accessibility following TCR stimulation. The latter was accompanied by nucleosome loss over the IL-2 promoter as was previously published by Shannons laboratory (Mol. Cell Biol. 25:3209, 2005). This loss was not observed in naive T cells even at the 48 hour time point. Finally, basal levels of cRel expression were higher in pre-activated T cells and greater levels were found sooner in the nucleus following TCR stimulation. Our current working model is that nucleosome disassembly facilitates the rapid initiation of IL-2 transcription in pre-activated T cells and that cRel signaling participates in this process. In naive CD4+ T cells the cRel component is rate-limiting and requires TNFR signalling to boost or sustain a high enough level to initiate IL-2 transcription.